Human granulocytic anaplasmosis (HGA) is a severe illness with a fatality rate of <5% that, like spotted fever, is caused by tick-borne intracellular bacteria, presenting a highly relevant, challenging opportunity to study host-vector-pathogen interactions. In this revised application we propose to build on key accomplishments from the prior funding period: 1) isolation of non-human-infectious Anaplasma phagocytophilum (Ap) from Camp Ripley, MN, ticks; 2) stable transformation of Ap and identification of genes required for growth in tick or human cells; 3) Identification of microvascular endothelial cells as a likely reservoir of infection; 4) demonstration of infection in multiple organs of mice; and, 5) whole genome transcription profiling of Ap during growth in human and tick cells, revealing host specific gene activity. These achievements will facilitate research during the next phase to further elucidate the events at the tick-mammal interface that shape pathogenesis of HGA. We hypothesize: 1) Whole genome comparison of Ap isolates that infect humans versus those that are pervasively found in ticks and wild animals at Camp Ripley will reveal genetic differences that underlie Ap pathogenicity. 2) Ap differentially regulates gene expression in a host cell specific manner, and temporally in a stage specific manner during infection. We will address these hypotheses under 4 specific aims: 1) We will sequence the genomes of Ap isolates from humans (from the upper Midwest and Northeast) and 5 from ticks and small mammals collected at Camp Ripley, and determine the differences between them and the sequenced Ap genome to correlate genome structure, gene content and infectivity for humans. 2) Whole genome expression arrays probed with Ap mRNA during specific steps in their life cycle (host cell adhesion, invasion, replication, exit) will identify key genes involved in these processes. Analysis of Ap development during distinct stages in human and tick cell culture will provide a detailed record of Ap gene activity during its life cycle. Under aim 3) we will create a library of Ap mutants using Himar1-mediated transformation of Ap and an inducible promoter system to obtain, screen and map transformants that display phenotypic changes. Conditionally fatal gene knock-outs can be obtained using the Himar1 transposon system with the tet repressor regulated promoter, and, in conjunction with alternating cell culture systems, will dramatically enhance recovery of transformants. By specifically disrupting Ap gene expression using insertional mutagenesis the essential processes which Ap performs to infect its different hosts can be sorted into common an host-specific genetic tools. Finally, we will integrate all data into the Artemis genome viewer/annotation tool to identify genomic sequence differences among isolates and construct an interrelated network of gene expression in Ap during specific life cycle phases. The proposed research will lead to a better understanding of Ap as an emerging human pathogen, providing new strategies for diagnosis, control and treatment. PUBLIC HEALTH RELEVANCE: Human granulocytotropic anaplasmosis (HGA) is an emerging tick-borne disease that, like Lyme disease, is transmitted by black-legged ticks (deer ticks) primarily in the Northeast and upper Midwest of the US, but mostly affects persons 60 years of age or older. The agent, Anaplasma phagocytophilum, causes sudden onset of high fever during the summer months requiring hospitalization for 40% of patients and has a fatal outcome in ~5% of clinical cases. The research proposed herein will reveal differences in the genetic code of A. phagocytophilum isolates that account for their ability to infect humans or animals, improve understanding of the epidemiology, identify crucial steps in cell invasion, replication and transmission that, when disrupted, could prevent progression of disease, and establish a novel suite of molecular tools to analyze the function of specific genes. Taken together, our research will continue to identify new leads on how to prevent and cure human anaplasmosis and similar diseases such as ehrlichiosis and spotted fevers.